Effects of Active-Center Reduction of Plant-Type Ferredoxin on Its Structure and Dynamics: Computational Analysis Using Molecular Dynamics Simulations.

"Plant-type" ferredoxins (Fds) in the thylakoid membranes of plants, algae, and cyanobacteria possess a single [2Fe-2S] cluster in active sites and mediate light-induced electron transfer from Photosystem I reaction centers to various Fd-dependent enzymes. Structural knowledge of plant-type Fds is relatively limited to static structures, and the detailed behavior of oxidized and reduced Fds has not been fully elucidated. It is important that the investigations of the effects of active-center reduction on the structures and dynamics for elucidating electron-transfer mechanisms. In this study, model systems of oxidized and reduced Fds were constructed from the high-resolution crystal structure of Chlamydomonas reinhardtii Fd1, and three 200 ns molecular dynamics simulations were performed for each system. The force field parameters of the oxidized and reduced active centers were independently obtained using quantum chemical calculations. There were no substantial differences in the global conformations of the oxidized and reduced forms. In contrast, active-center reduction affected the hydrogen-bond network and compactness of the surrounding residues, leading to the increased flexibility of the side chain of Phe61, which is essential for the interaction between Fd and the target protein. These computational results will provide insight into the electron-transfer mechanisms in the Fds.

Chemical synthesis of ferredoxin with 4 selenocysteine residues using a segment condensation method.

Ferredoxin (Fd) is an electron carrier protein containing a [2Fe-2S] cluster. In this paper, we synthesized Se-Fd, in which four Cys residues coordinated to the cluster are substituted to selenocysteine. After the one-pot segment coupling by the thioester method, followed by deprotection and cluster loading, the desired Se-Fd was successfully obtained.

X-ray dose-dependent structural changes of the [2Fe-2S] ferredoxin from Chlamydomonas reinhardtii.

Plant-type ferredoxin (Fd) is an electron transfer protein in chloroplast. Redox-dependent structural change of Fd controls its association with and dissociation from Fd-dependent enzymes. Among many X-ray structures of oxidized Fd have been reported so far, very likely a given number of them was partially reduced by strong X-ray. To understand the precise structural change between reduced and oxidized Fd, it is important to know whether the crystals of oxidized Fd may or may not be reduced during the X-ray experiment. We prepared the thin plate-shaped Fd crystals from Chlamydomonas reinhardtii and monitored its absorption spectra during experiment. Absorption spectra of oxidized Fd crystals were clearly changed to that of reduced form in an X-ray dose-dependent manner. In another independent experiment, the X-ray diffraction images obtained from different parts of one single crystal were sorted and merged to form two datasets with low and high X-ray doses. An Fo-Fo map calculated from the two datasets showed that X-ray reduction causes a small displacement of the iron atoms in the [2Fe-2S] cluster. Both our spectroscopic and crystallographic studies confirm X-ray dose-dependent reduction of Fd, and suggest a structural basis for its initial reduction step especially in the core of the cluster.

Stabilized pyrrolyl iodonium salts and metal-free oxidative cross-coupling.

Pyrrole-aryl derivatives are important due to their unique biological activities in medicinal chemistry. We now report a new oxidative biaryl coupling for pyrroles and indoles toward various arenes using a hypervalent iodine reagent and an appropriate stabilizer for pyrrolyl iodonium intermediates. The reactions readily provide a variety of desired coupling products in good yields.

Temporal impulse response function of the visual system estimated from ocular following responses in humans.

Early visual processing functions as a set of spatiotemporal image filters. Our ability to sense changes in retinal images is determined by these filters along the temporal axis. In this study, we developed a paradigm to identify the kernel of the temporal filters based on ocular following responses (OFRs) to two-frame apparent motion stimuli. We first conducted two experiments to acquire fundamental data. In the first experiment, in which a quarter wavelength step of a sinusoidal grating was presented with various inter-stimulus intervals (ISIs), we found that OFRs were reversed by the ISI, which is consistent with previous findings. In the second experiment, a quarter wavelength step of a sinusoidal grating was applied with various durations of the initial image frame (motion onset delays; MODs); we found that longer exposure to the initial image reduced OFRs. Parameters of motion energy model involving temporal filters were optimized so that the model could reproduce the dependence of OFRs on ISIs and MODs. We were then able to successfully obtain quantitative estimates of the biphasic temporal filters with optimal frequencies in 6-8Hz. This method is completely objective and will thus be applicable to a wide range of human subjects and model animals.

Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages.

It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterized the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.

Single-electron-transfer (SET)-induced oxidative biaryl coupling by polyalkoxybenzene-derived diaryliodonium(III) salts.

Metal-free oxidative C-C coupling by using polyalkoxybenzene-derived diaryliodonium(III) salts as both the oxidant and aryl source has been developed. These salts can induce single-electron-transfer (SET) oxidation to yield electron-rich arenes and subsequently transfer the polyalkoxyphenyl group into in situ generated aromatic radical cations to produce biaryl products. The reaction is promoted by a Lewis acid that activates the iodonium salts. It has been revealed that the reactivity of the salts under acidic conditions is quite different to their known behavior under basic conditions. The reactivity preference of a series of iodonium salts in the SET oxidation and their ligand transfer abilities have been systematically investigated and the results are summarized in this report.

Unique structural properties of 2,4,6-tri-tert-butylanilide: isomerization and switching between separable amide rotamers through the reaction of anilide enolates.

Herein, we report a unique structural property of 2,4,6-tri-tert-butylanilide, which can be separated into its amide rotamers at room temperature. Interconversion between the rotamers of anilide enolates occurs readily at room temperature and their reaction with electrophiles gives mixtures of the rotamers in a ratio that depends on the reactivity of the corresponding electrophile. That is, the reaction of the 2,4,6-tri-tert-butylacetanilide enolate with reactive electrophiles, such as allyl bromide or protic acids, gives mixtures of the anilide rotamers in which the E rotamer is the major component, whereas less-reactive electrophiles, such as 1-bromopropane and 2-iodopropane, yield mixtures of the rotamers in which the Z rotamer is the major component. The rotameric ratio of the product is also strongly dependent on the reactivity of the anilide enolate. Switching between the anilide rotamers can be achieved through protonation of a less-reactive enolate by a less-reactive protic acid and thermal isomerization of the anilide.

Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development.

The transcription factors Nanog and Gata6 are critical to specify the epiblast versus primitive endoderm (PrE) lineages. However, little is known about the mechanisms that regulate the protein stability and activity of these factors in the developing embryo. Here we uncover an early developmental function for the Polycomb group member Bmi1 in supporting PrE lineage formation through Gata6 protein stabilization. We show that Bmi1 is enriched in the extraembryonic (endoderm [XEN] and trophectodermal stem [TS]) compartment and repressed by Nanog in pluripotent embryonic stem (ES) cells. In vivo, Bmi1 overlaps with the nascent Gata6 and Nanog protein from the eight-cell stage onward before it preferentially cosegregates with Gata6 in PrE progenitors. Mechanistically, we demonstrate that Bmi1 interacts with Gata6 in a Ring finger-dependent manner to confer protection against Gata6 ubiquitination and proteasomal degradation. A direct role for Bmi1 in cell fate allocation is established by loss-of-function experiments in chimeric embryoid bodies. We thus propose a novel regulatory pathway by which Bmi1 action on Gata6 stability could alter the balance between Gata6 and Nanog protein levels to introduce a bias toward a PrE identity in a cell-autonomous manner.

Active role of small non-coding RNAs derived from SINE/B1 retrotransposon during early mouse development.

Small RNAs derived from repetitive sequences appear to play essential roles in mammalian gametogenesis and early development. In this study we focused on the short interspersed nucleotide element B1 (SINE/B1) small RNAs, which were zygotically expressed in pre-implantation mouse embryos; and we investigated whether the SINE/B1 small RNAs played an active role in gene silencing during early mouse development. The results indicated that the level of silencing activity involving the SINE/B1 small RNAs as mediators was significantly reduced in Dicer-knockdown mouse embryos. In addition, when the SINE/B1 small RNAs were mapped to a full-length SINE/B1 sequence, phase-distribution of the small RNAs appeared, suggesting possible enzymatic involvement. Therefore, our present study suggested that the zygotically expressed SINE/B1 small RNAs in pre-implantation mouse embryos contain active small RNAs, which were presumably processed by Dicer and involved in gene silencing.

Notable structural property of 2,4,6-tri-tert-butylanilide enolates: interconversion between the rotamers and their reactivity.

Interconversion between the separable 2,4,6-tri-tert-butylanilide rotamers was found to easily occur through formation of the lithium enolate. Protonation of the anilide enolate gave the anilide rotamer mixture of E-major. On the other hand, reactions of lithium enolate prepared from 2,4,6-tri-tert-butylpropionanilide with alkyl bromides preferentially afforded a Z-rotamer of alkylated products.

Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development.

Recent studies showed that small interfering RNAs (siRNAs) and Piwi-interacting RNA (piRNA) in mammalian germ cells play important roles in retrotransposon silencing and gametogenesis. However, subsequent contribution of those small RNAs to early mammalian development remains poorly understood. We investigated the expression profiles of small RNAs in mouse metaphase II oocytes, 8-16-cell stage embryos, blastocysts and the pluripotent inner cell mass (ICM) using high-throughput pyrosequencing. Here, we show that during pre-implantation development a major small RNA class changes from retrotransposon-derived small RNAs containing siRNAs and piRNAs to zygotically synthesized microRNAs (miRNAs). Some siRNAs and piRNAs are transiently upregulated and directed against specific retrotransposon classes. We also identified miRNAs expression profiles characteristic of the ICM and trophectoderm (TE) cells. Taken together, our current study reveals a major reprogramming of functional small RNAs during early mouse development from oocyte to blastocyst.

Variation of gene silencing involving endogenous microRNA in mammalian cells.

MicroRNAs (miRNAs) are small noncoding RNA and play a role in gene expression regulation by inhibiting translation of their target messenger RNAs (mRNAs). In this study, we investigated the effects of endogenous let-7 miRNA on the expression of target genes in various mammalian cells by means of two types of reporter plasmids possessing target sequences for let-7: one carries perfectly matched target sequence for let-7 in the 3'-untranslated region of the luciferase reporter gene to monitor RNA interference (RNAi) activity and the other has three bulged binding sites for let-7 to monitor translation-inhibition activity. The results indicate that different cells have different levels of gene silencing against the target reporter genes. The data presented here suggest that not only microRNA level but also target transcript level likely participate in the generation of a variety of gene silencing.

Enhancement of allele discrimination by introduction of nucleotide mismatches into siRNA in allele-specific gene silencing by RNAi.

Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs) against mutant alleles of the human Prion Protein (PRNP) gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the 'seed region' of microRNAs. Due to the essential role of the 'seed region' of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs), of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3'-ends of sense-strand siRNA elements, which possibly increase the assembly of antisense-strand (guide) siRNAs into RNA-induced silencing complexes (RISCs), may enhance ASP-RNAi in the case of inert siRNA duplexes. Therefore, the data presented here suggest that structural modification of functional portions of an siRNA duplex by base substitution could greatly influence allele discrimination and gene silencing, thereby contributing to enhancement of ASP-RNAi.

Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles.

Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically suppressing the expression of alleles associated with disease. To realize such allele-specific RNAi (ASPRNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital, but is also difficult. Here, we show ASP-RNAi against the Swedish- and London-type amyloid precursor protein (APP) variants related to familial Alzheimer's disease using two reporter alleles encoding the Photinus and Renilla luciferase genes and carrying mutant and wild-type allelic sequences in their 3'-untranslated regions. We examined the effects of siRNA duplexes against the mutant alleles in allele-specific gene silencing and off-target silencing against the wild-type allele under heterozygous conditions, which were generated by cotransfecting the reporter alleles and siRNA duplexes into cultured human cells. Consistently, the siRNA duplexes determined to confer ASP-RNAi also inhibited the expression of the bona fide mutant APP and the production of either amyloid beta 40- or 42-peptide in Cos-7 cells expressing both the full-length Swedish- and wild-type APP alleles. The present data suggest that the system with reporter alleles may permit the preclinical assessment of siRNA duplexes conferring ASP-RNAi, and thus contribute to the design and selection of the most suitable of such siRNA duplexes.

Influence of assembly of siRNA elements into RNA-induced silencing complex by fork-siRNA duplex carrying nucleotide mismatches at the 3'- or 5'-end of the sense-stranded siRNA element.

RNA interference (RNAi) is a powerful method for suppressing the expression of a gene of interest, and can be induced by 21-25 nucleotide small interfering RNA (siRNA) duplexes homologous to the silenced gene, which function as sequence-specific RNAi mediators in RNA-induced silencing complexes (RISCs). In the previous study, it was shown that fork-siRNA duplexes, whose sense-stranded siRNA elements carried a few nucleotide mismatches at the 3'-ends against the antisense-stranded siRNA elements, could enhance RNAi activity more than conventional siRNA duplexes in cultured mammalian cells. In this study, we further characterized fork-siRNA duplexes using reporter plasmids carrying target sequences complementary to the sense- or antisense-stranded siRNA elements in the untranslated region of Renilla luciferase. The data presented here suggest that nucleotide mismatches at either the 3'- or 5'-end of the sense-stranded siRNA elements in fork-siRNA duplexes could influence assembly of not only the antisense-stranded siRNA elements but also the sense-stranded elements into RISCs. In addition, we further suggest the possibility that there could be a positional effect of siRNA duplex on RNAi activity.